Esters of {60 -aminobenzylpenicillin

ABSTRACT

The invention comprises antibiotically active new esters of Alpha -aminobenzylpenicillin of the formula: IN WHICH THE ASTERISK INDICATES AN ASYMMETRIC CARBON ATOM; N IS AN INTEGER FROM 0 TO 5; AND A is an unsubstituted or substituted aliphatic, alicyclic, aromatic, or heterocyclic radical; the salts of said esters with pharmaceutically acceptable acids; and methods of preparing the esters and salts. This invention relates to a group of new esters of Alpha aminobenzylpenicillin, these esters having the formula IN WHICH THE ASTERISK INDICATES AN ASYMMETRIC CARBON ATOM; N IS AN INTEGER FROM 0 TO 5; AND A is an unsubstituted or substituted aliphatic, alicyclic, aromatic, or heterocyclic radical, to salts of these esters with pharmaceutically acceptable acids, and to methods of preparing these new esters. More particularly, A may represent an aliphatic hydrocarbon radical in which the carbon chain can be straight or branched, saturated or unsaturated, having from one to six carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, sec. and tert. butyl, pentyl, hexyl, and the like; an alicyclic, carbocyclic radical having from three to 10 carbon atoms as ring members, and in which the ring or rings may be saturated or may contain one or two double bonds depending on the number of carbon atoms, such as cyclopentyl, cyclohexyl, 1-adamantyl, 1-bicyclo(2.2.2)octyl, cyclopentenyl, cyclohexenyl, in which the double bond may be placed in the 2,3- or 3,4-position, and the like; an aromatic radical such as a monocyclic carbocyclic aryl radical, e.g., a phenyl or substituted phenyl radical, a bicyclic carbocyclic aryl radical, e.g. a 1- or 2napthyl or substituted naphthyl radical; a heterocyclic aryl radical, which may contain from five to 10 atoms as ring members, such as pyridyl, pyrazinyl, pyrimidyl, thienyl, furyl, or quinolyl, in which the hetero atom may be in any of the available positions, and which may further have substituents in one or more of the remaining positions. As already mentioned, the radical A may have further substituents, such as lower alkyl, e.g., methyl, ethyl, propyl, isopropyl, butyl and the like; lower alkoxy, e.g., methoxy, ethoxy and the like; lower alkylthio, e.g., methylmercapto or ethylmercapto; halo-lower alkyl, e.g., mono-, di- or trifluoromethyl, mono-, dior trichloromethyl or the ethyl homologues and the corresponding bromo derivatives; halogens, e.g., fluorine, bromine or chlorine; or nitro groups. The substituents may be placed in any possible positions. Salts of the new esters may be formed with inorganic acids, e.g., hydrochloric, hydrobromic, hydriodic acid and sulphuric acid and the like; and organic acids such as citric, tartaric, maleic acid and the like. Due to the asymmetric carbon atom in the side chain of the compounds of formula (I), these compounds exist in two epimeric forms, and the invention comprises both of the epimeric forms as well as mixtures thereof. The form, in which the compounds are obtained, depends on which of the epimeric starting materials and which coupling methods are used. The mixtures of the epimeric foRms may be separated by fractional crystallization or other known methods. It is known that the acid-resistant Alpha aminobenzylpenicillin is a broad-spectrum antibiotic with a widespread use. It is a disadcantage, however, that when orally administered, Alpha -aminobenzylpenicillin is insufficiently absorbed in the organism, and it is one object of the present invention to provide new antibiotically active derivatives of Alpha -aminobenzylpenicillin which, with a view to adequate absorption, distribution in the organism, and the like factors, are superior to Alpha -aminobenzylpenicillin. Acyloxymethyl esters of some penicillins are known, for instance from Dutch Pat. application No. 6,405,891, from which it appears that by oral administration such esters are practically not absorbed. An exception is the acetoxymethyl ester of benzylpenicillin, which to some degree is absorbed, giving rise to low but prolonged blood concentrations. It is therefore surprising that the compounds of the invention by oral administration give rise to extremely high concentrations of Alpha -aminobenzylpenicillin in blood and tissues due to an efficient absorption combined with a rapid hydrolysis in the organism. Table I below shows that the concentrations obtained in the blood and various organs, such as liver, lungs, kidneys and spleen, are considerably higher after administration of the pivaloyloxymethyl ester of D(-)- Alpha -aminobenzylpenicillin than after administration of a corresponding dose of D(-)- Alpha -aminobenzylpenicillin.

United States Patent 1151 3,697,507

Frederiksen et a]. 1 Oct. 10, 1972 ESTERS 0F 1- [56] References CitedAMINOBENZYLPENICILLIN UNITED STATES PATENTS [72] 3 g fi gl o f'2,985,648 5/1961 Doyle et a1. ..260/239.l b 5" 1 k aemse 3,228,930l/1966 Sjpberg etal ..260/239.l 0 3,250,679 5/1966 Jansen et al...260/239.1 [73] Assignee: Lovens kemiske FabrikProduktionsaktieselskab, Ballerup, Denmark Primary 11 2 Rluo tt 22Filed: Sept. 26, 1968 A Omey Stowe i [21] 'Appl. No.: 762,987 ABSTRACTThe invention comprises antibiotically active new [30] ForeignApplication p i i Data esters of a-aminobenzylpenicillin of the formula:

Sept. 29, 1967 Great Britain ..44,535/67 Oct. 5, 1967 Great Britain..45,600/67 Oct. 23, 1967 Great Britain ..48,127/67 '(|3H'CNH'CHC O\Nov. 10, 1967 Great Britain ..5l,358/67 NHz i I l 0m Dec. 6, 1967 GreatBritain ..55,489/67 0 N OO O Jan. 3, 1968 Great Britain ..499/68 (1)March 22, 1968 Great Britain ..l4,04l/68 t, ..1.. Ma March 29, I968Great Britain 15,312/68 in which the asterisk indicates an asymmetriccarbon [52] US. Cl ..260/239.l, 424/271 atom; n is an integer from 0 to5; and A is an unsub- [51] Int. Cl. ..C07d 99/16 u d or su st tut daliphati alicyclic, aromatic, or [58] Field of Search ..260/239.1heterocyclic radical; the Salts of Said esters with P maceuticallyacceptable acids; and methods of preparing the esters and salts.

7 Claims, No Drawings ESTERS OF a-AMINOBENZYLPENICILLIN This inventionrelates to a group of new esters of aaminobenzylpenicillin, these estershaving the formula NH: CH3

o-N-CH-C 01120 C ()(CHQ A.

in which the asterisk indicates an asymmetric carbon atom; n is aninteger from 0 to 5; and A is an unsubstituted or substituted aliphatic,alicyclic, aromatic, or heterocyclic radical, to salts of these esterswith pharmaceutically acceptable acids, and to methods of preparingthese new esters.

More particularly, A may represent an aliphatic hydrocarbon radical. inwhich the carbon chain can be straight or branched, saturated orunsaturated, having from one to six carbon atoms, such as methyl, ethyl,propyl, isopropyl, butyl, sec. and tert. butyl, pentyl, hexyl, and thelike; an alicyclic, carbocyclic radical having from three to carbonatoms as ring members, and in which the ring or rings may be saturatedor may contain one or two double bonds depending on the number of carbonatoms, such as cyclopentyl, cyclohexyl, l-adamantyl,1-bicyclo(2.2.2)octyl, cyclopentenyl, cyclohexenyl, in which the doublebond may be placed in the 2,3- or 3,4-position, and the like; anaromatic radical such as a monocyclic carbocyclic aryl radical, e.g., aphenyl or substituted phenyl radical, a bicyclic carbocyclic arylradical, e.g. a 1- or 2napthyl or substituted naphthyl radical; aheterocyclic aryl radical, which may contain from five to 10 atoms asring members, such as pyridyl, pyrazinyl, pyrimidyl, thienyl,

furyl, or quinolyl, in which the hetero atom may be in any of theavailable positions, and which may further have substituents in one ormore of the remaining positions. As already mentioned, the radical A mayhave further substituents, such as lower alkyl, e.g., methyl, ethyl,propyl, isopropyl, butyl and the like; lower alkoxy, e.g., methoxy,ethoxy and the like; lower alkylthio, e.g., methylmercapto orethylmercapto; halo-lower alkyl, e.g., mono-, dior trifluoromethyl,mono, dior trichloromethyl or the ethyl homologues and the correspondingbromo derivatives; halogens, e.g., fluorine, bromine or chlorine; ornitro groups. The substituents may be placed in any possible positions.Salts of the new esters may be formed with inorganic acids, e.g.,hydrochloric, hydrobromic, hydriodic acid and sulphuric acid and thelike; and organic acids such as citric, tartaric, maleic acid and thelike.

Due to the asymmetric carbon atom in the side chain of the compounds offormula (1), these compounds exist in two epimeric forms, and theinvention com prises both of the epimeric forms as well as mixturesthereof. The form, in which the compounds are obtained, depends on whichof the epimeric starting materials and which coupling methods are used.The mixtures of the epimeric forms may be separated by fractionalcrystallization or other known methods.

It is known that the acid-resistant a-aminobenzylpenicillin is abroad-spectrum antibiotic with a widespread use. It is a disadcantage,however, that when orally administered, a-aminobenzylpenicillin isinsufficiently absorbed in the organism, and it is one object of thepresent invention to provide new antibiotically active derivatives ofa-aminobenzylpenicillin which, with a view to adequate absorption,distribution in the organism, and the like factors, are superior toa-aminobenzylpenicillin.

Acyloxymethyl esters of some penicillins are known, for instance fromDutch Pat. application No. 6,405,891 from which it appears that by oraladministration such esters are practically not absorbed. An exception isthe acetoxymethyl ester .of benzylpenicillin,

' which to some degree is absorbed, giving rise to low but TABLE I.

Concentrations of D(-)-a-aminobenzylpenicillin in blood and organs'fromrats after oral administration of A. D(-)-a-aminobenzylpenicillinmg/kg.) B. Pivaloyloxymethyl D(-)-a-aminobenzylpenicillinate, HC] (143mg./kg.) equivalent to 100 mg. of D(- )-a-aminobenzylpenicillin.

pg. per ml. or per g. wet weight of tissues Organ V2 hour 1 hour 2 hoursA B A B A B Blood 5. 1 1 21 4.6 14 1.4 2.7

Liver 20 50 16 37 Lungs 1.3 7.9 1.1 6.5 0.56 2.0 Kidneys 21 84 25 67 9.625 Spleen 0.65 2.4 0.60 2.6 0.34 0.93

Table II below shows the difference in the serum concentrations of.benzylpenicillin and D(-)-aaminobenzylpenicillin, respectively, in dogsfollowing single oral doses of the acetoxymethyl esters of these twopenicillins equivalent to 20 mg./kg. body weight ofa-aminobenzylpenicillin.

TABLE II.

Serum concentrations in ugJml.

Hours after dose A l 2 4 5 Acetoxymethyl ester of benzylpenicillin 0.900.7] 0.36 0.1 l

Acetoxymethyl ester of D(-)-a-amin oben;ylpenicillin 10.45 7.30 1.230.32

+ Dutch Patent Application No. 6,405,981

The same effect is also seen in man.

In Table Ila below, the serum concentrations ofD(-)-a-aminobenzylpenicillin after oral administration to normal humanvolunteers of 715 mg. of the pivaloylox- TABLE Ila.

Serum concentrations in man in pg/ml.

Hours after administration l 2 When the esters of formula I are exposedto the influence of enzymes present in the body fluids, or to enzymesproduced by microorganisms, e.g., pathogenic micro-organisms, they arehydrolysed hydrolysed to aminobenzylpenicillin. aminobenzylpenicillin.This hydrolysis is an important feature of the compounds of theinvention. It is assumed that the first step consists in an enzymatichydrolysis by non-specific esterase to the corresponding hydroxymethylesters of a-aminobenzylpenicillin which subsequently decomposespontaneously to tat-aminobenzylpenicillin.

The compounds of formula (I) are well tolerated compounds which maypreferably be administered orally either as such or in form of one oftheir salts, and may be mixedup with a solid carrier and/or auxiliaryagents.

In such compositions, the proportion of therapeutically active materialto carrier substanceand auxiliary agent can vary between 1 percent and95 percent. The compositions can either be worked up to pharmaceuticalforms of presentation, such as tablets, pills or dragees, or can befilled in medical containers such as capsules, or as far as mixtures areconcerned filled into bottles.

Pharmaceutical organic or inorganic solid or liquid carriers suitablefor oral, enteral or topical administration can be used to make up thecomposition. Gelatine, lactose, starch, magnesium stearate, talc,vegetable and animal fats and oils, gums, polyalkylene glycol, or otherknown carriers for medicaments are all suitable as carriers.

The preferred salt of the esters is the hydrochloride, but salts withother inorganic or organic acids including antibiotically active acidsmay be used, e.g., the phosphate, the acetate, thephenoxymethylpenicillin salt, and the like. Furthermore, thecompositions may contain other pharmaceutically active components whichcan appropriately be administered together with the ester in thetreatment of infectious diseases, such as other suitable antibiotics.

The favorable high blood levels obtained after oral administration of asingle dose of two of the compounds ofthe invention equivalent to 250mg. of aaminobenzylpenicillin appear from table III, in which thefigures show the serum concentrations in ugJml. of the two estersmentioned compared with the aaminobenzylpenicillin itself.

TABLE III.

1/2 h 1 h 2 h 4 h Aminobenzyl- 4 penicillin 0.7 L6 1.8 0.8 AcetoxymethylD(-)- aaminobenzylpenicillin 4.5 3.3 1.7 0.6 PivaloyloxymethylD(-)-aaminobenzylpenicillin 5.0 5.9 2.2 0.5

The present invention also includes methods for the preparation of thenew esters and their salts. Owing to the presence of the 01 amino groupof the side chain and the hydrolyzable ester group of the compounds ofthe invention, certain considerations have to be taken in thepreparation.

One method comprises a firststep in which an a-substitutedbenzylpenicillin derivative of the formula (II is reacted with acompound of formula (III) according to the followingscheme:

In these formulas, n and A are as defined hereinbefore; R is an aminogroup, or a substituted amino group Z-NH, or a group in which can beconverted into an amino group, such as an azido or nitro group, or ahalogen atom; COOY and X--CH represent radicals capable of reacting witheach other to form the COO-CH grouping. As Examples of such radicals, Ymay be hydrogen or an alkali metal or a tertiary ammonium group and Xmay be a halogen atom, preferably chlorine or bromine, an acyloxyradical having from one to 16 carbon atoms, or an alkylsulphonyloxy orarylsulphonyloxy radical. The compounds formed by the above-mentionedreaction are esters of a-R-substituted benzylpenicillins (IV). When R isNI-I formula (IV) represents the compounds of the invenabove, formula(IV) represents interesting intermediates in the synthesis of thecompound of the invention, and as such constitute a part of thisinvention. As a common characteristic of the substituent R, it can besaid that it is selected from groups which after the above reaction arecapable of being converted into an amino group by means of methods mildenough to avoid a cleavage of the molecule at the ester group or at thelactam ring. In particular, the substituent R may have the formula Z-NHwhere Z is a benzoyloxycarbonyl radical, a p-halo-, p-nitro-, orp-methoxybenzyloxycarbonyl radical, a B, B, B-trichloroethyloxycarbonylor an allyloxycarbonyl radical; or Z is a sulphur-containing radical,such as tritylsulphenyl radical, or an arylsulphenyl radical, e.g., ano-nitrophenylsulphenyl radical; 2 may also be a triphenylmethyl (alsocalled trityl) radical, a tertiary butoxycarbonyl radical, or a radicalobtained by reacting the free amino group with a ,B-dicarbonyl compoundsuch as acetylacetone, acetoacetic esters or benzoyl-acetone to formenamines or Schiff bases. In general, any group represented by Z, whichcan be split off be reduction, by mild acid hydrolysis or by other mildreactions known per se will be suitable, since experiments have shown,that the esters of formula (I) formed by the reaction in question arestable under conditions. As examples of R substituents which can beconverted into an amino group mention can be made of the azido group,the nitro group and halogen atoms, for instance a bromine atom. Thestarting compounds of formula (II), in which R is different from NI-Iare known as intermediates in the synthesis of a-aminobenzylpenicillin.They exist in two epimeric forms. If the starting materials are preparedin form of the D or the L epimers, the corresponding epimeric form ofthe compounds of the invention will be obtained. If, on the other hand,a mixture of the epimeric forms of the starting compound is used, amixture is obtained. This mixture can be separated in the two epimers,for instance, by fractional crystallization.

The methods of preparing the starting substances of formula (II) arestandard procedures employed in peptide chemistry and include, forinstance, the conversion of the phenylacetic acid to the R-substitutedphenylacetic acid, R having the meaning given before, followed by areaction between a reactive derivative of this intermediate and6-aminopenicillanic acid in which the amino group may be free orsubstituted, for instance with a trimethylsilyl radical. Some of thestarting substances of formula (II) can also be prepared fromaaminobenzylpenicillin or salts thereof.

Some of the starting compounds of formula (III) are known compounds, thepreparation of which is described in the literature. Others are new, butcan be prepared in the same way as the known, using methods which arestandard procedures for this type of compounds.

Among such methods may be mentioned the reaction of an acid halide withparaformaldehyde (as described in e.g..l.A.C.S. 43, 660 (1921) or thehalogenation of methyl esters (as described in, e.g.,

dioxane, tetrahydrofuran, methylene chloride and dimethylformamide. Thereaction products are crystalline or oily products, which can be used inthe next step without further purification. By repeatedreprecipitations, the oily products can be converted into crystalline oramorphous powders.

The subsequent reaction step (IV-I), by which the group R is convertedinto an amino group, may be effected by different methods known frompeptide synthesis, depending on what R stands for.

Catalytic hydrogenation will be preferred with R has the formula ZNH,and Z stands for benzyloxycarbonyl and related derivatives thereof, andfor trityl. This hydrogenation is preferably performed at roomtemperature and at atmospheric or slightly elevated pressure in asolvent which may be a non-reducible organic solvent, or a mixturethereof with water. The preferred catalysts are noble metal catalysts,such as palladium or platinum, or Raney-Nickel, but other catalysts canbe used as well. Electrolytic reduction can also be used in these cases.When Z stands for a [3,3,3- trichloroethyloxycarbonyl group, a reductionwith Zn in acetic acid is preferable. A mild acid hydrolysis ispreferred in the case where Z stands for a sulphur-containing radical,an enamine or a Schiff base, for instance by means of a dilute solutionof hydrogen chloride in aqueous acetone at a pH of about 2. A tre atmentwith formic acid at room temperature is especially suitable for theremoval of Z, when Z is a tertiary butoxycarbonyl radical. Also knownfrom the literature is the removal of the o-nitriphenylsulphenyl radicalinvolving a nucleophilic attack on the sulphur atom of the sulphenamidegroup, the best yield in the present case being obtained with sodium orpotassium iodide, sodium thiosulphate, sodium hydrogen sulphide, sodiu'mdithionite, or potassiumthiocyanate. Other sulphenamide radicals can besplit off in the same way. If R is an azido or nitro group, or a halogenatom, especially a bromine atom, these may be transformed into the freeamino group in known manner, the azido and the nitro group by acatalytic hydrogenation with a noble metal catalyst, or withRaney-Nickel, or by an electrolytic reduction, and the halogen atom byan amination, for instance with hexamethylenetetramine.

Another suitable method for the preparation of the compounds of thepresent invention comprises the reaction of a reactive derivative of ana-substituted phenylacetic acid of formula (V) with an ester of 6-aminopenicillanic acid of formula (VI) whereby a product of the aboveformula (IV) is obtained according to the following scheme:

S CH3 an acid chloride or bromide; an anhydride; a mixed anhydride withan alkyl-carbonic acid, such as isobutylcarbonic acid; a carboxylicacid; an inorganic acid or a sulphonicacid; or a radical obtained byreacting the asubstituted phenylacetic acid with a carbodiimide, orN,N'-carbonyldiimidazole, or a similarly functioning compound; X can behydrogen, or a trialkylsilyl group, the alkyl having not more than fivecarbon atoms.v

The reaction can be performed in an organic solvent or in a mixturethereof with water either at a low temperature or at slightly elevatedtemperature. Suitable solvents are methylene chloride, chloroform, ethylacetate, acetone, dimethylformamide, or dimethylacetamide, ether,tetrahydrofuran, dioxane, or similar inert solvents.

The reaction products are isolated in conventional manner, e.g., byreprecipitation or by removalof the solvent followed byrecrystallization from a solvent. The starting compounds of formula (V)are known compounds which can be prepared by standard methods known fromthe peptide chemistry.

The compounds of formula (VI) are new compounds, which are interestingintermediates in the synthesis of the compounds of formula (I) and assuch constitute a part of this invention. They can be prepared by thetreatment of 6-amino-penicillanic acid in the form of a salt, such as analkali metal salt or the .triethylammonium salt, with a halomethyl esterof the formula R CH -OCO(CI-I,),,A (VII) in which R is a halogen,preferably a chlorine or bromine atom, or a sulphonyloxy radical, suchas methanesulphonyloxy or toluenesulphonyloxy radical, and n and A areas defined above. The 6-amino-penicillanic acid may be used as such, orthe 6-amino group may be protected during the esterification process.Only protecting radicals, which can be easily removed without causingany cleavage of the lactam ring or the ester group, are suitable in thiscase, for instance triphenylmethyl or trimethylsilyl radicals. Thereaction is performed in an inert organic solvent such as acetone,dimethylformamide, or methylene chloride, and at or below roomtemperature, or at slightly elevated temperatures. When the amino grouphas been protected, the removal of the protecting group can be performedby different methods, such as hydrogenolysis or hydrolysis under neutralor acidic conditions which do not attack the B-lactam ring and the estergroup. The reaction products of formula (VI) X'=I-1) are convenientlyisolated as their acid addition salts with, for instance,ptoluenesulphonic acid or other inorganic or organic acids such assulphuric, phosphoric, hydrochloric, acetic, maleic, tartaric and thelike acids.

In another embodiment of the process, the compounds of formula (VI) canbe prepared by esterification of any of the industrially accessiblepenicillins, or preferably their salts, with a compound of theaforementioned formula (VII) under similar conditions as alreadydescribed, whereafter the side chain of the resulting penicillin esteris split off to yield the 6-amino penicillanic ester of formula (VI), ora salt thereof.

The cleavage of the amide bond can be performed by a modification of theprocedure described in Belgian Pat. specification No. 698.596 byreacting the 6- acylamino-penicillanic acid ester with an acid halide inthe presence of an acid-binding agent, such s quinoline or pyridine, andthe like. The preferred halide is, however, phosphorus pentachloride,because the reaction in this case can be performed at low temperatureincreasing the stability of the intermediate formed, which presumably isan imino halide. The reaction can be performed in different solvents,but the preferred ones are chloroform and methylene chloride.

The intermediate is not isolated, but is treated with an excess of aprimary alcohol to form an imino ether. The reaction temperature and thereaction time depend on the alcohol used; in most cases temperaturesfrom -20 C to +20 C will be convenient.

The imino ether is not isolated, but subjected to an acid alcoholysis orhydrolysis, whereby the C=N bond is cleft to yield the corresponding6-amino-penicillanic ester of formula (VI). It is surprising that thelactam ring and the acyloxymethyl ester grouping is sufficiently stableunder these conditions. By the generally used methods, the esters of6-amino-penicillanic acid can be isolated from the reaction mixture assuch or in the form of a salt with an inorganic or organic acid such asthe hydrochloride or tosylate. 7

The invention will now be illustrated by the following non-limitingExamples:

EXAMPLE 1 A. Acetoxymethyl a-Azido-Benzylpenicillinate A mixture ofpotassium a-azido-benzylpenicillinate (2 g.), potassium bicarbonate (0.5g.), acetoxymethyl bromide (1.5 ml.) and acetone (2 percent water)(20ml.) was refluxed for 1 hour. After cooling, the suspension was filteredand the filtrate evaporated in vacuo. The oily residue was washedrepeatedly by decantation with petroleum ether to yield the desiredester as a gum (2.5 g.) which did not crystallize. B. Acetoxymethyla-Amino-Benzylpenicillinate Hydrochloride To a solution of acetoxymethyla-azidobenzylpenicillinate (2 g.) in ethyl acetate (20 ml.) was added asolution of H PO (980 mg.) and KH PO (1360 mg.) in water (20 ml.). 10percent palladium on carbon catalyst (2 g.) was added, and the resultingmixture was shaken in a hydrogen atmosphere for 2 hours. The catalystwas filtered off, and the phases were separated. The aqueous phase waswashed with ether, neutralized (pH 7.5) with aqueous sodium bicarbonate,and extracted several times with ethyl acetate. The combined extractswere washed with water, dried, and evaporated in vacuo. The oily residuewas suspended in ethanol (10 ml.), and 2N ethanolic hydrogen chloride(2.5 ml.) was added with stirring. Addition of ether to the resultingsolution precipitated the hydrochloride of acetoxymethyla-amino-benzylpenicillinate, which was filtered off, washed with ether,and dried. The product thus obtained was a colorless amorphous powder,readily soluble in water, methanol and ethanol, but sparingly soluble inether and petroleum ether. The purity was determined iodometrically tobe 86 per cent. The I.R. spectrum (KBr) contains strong bands at:

692 crn. 977 cm. 1020 cm." 1195 cm. 1300 cm." 1370 cm. 1460 cm." 1500cm. 1500 cm. I687 cm. 1765 cm.

The free base was precipitated from an aqueous solution of thehydrochloride by the addition of aqueous sodium bicarbonate, asacolorless amorphous powder. The N.M.R.-spectrum (CDCl of this productshows characteristic signals at 6 7.32(s), 5.76(d), 5.53,

4.52, 4.43, 2.08(s), l.62(s), and l.50(s), TMS being used as internalreference.

EXAMPLE 2 D(-)-a-Amino-Benzylpenicillin Acetoxymethyl Ester,Hydrochloride D(-)-a-aminobenzylpenicillin (3.5 g.) and triethylamine(1.42 ml.) were mixed with acetone containing 1 per cent of water (70ml.). To the resulting solution was added potassium bicarbonate (l g.)and bromomethyl acetate (1.75 ml.), and the mixture was stirred at roomtemperature for 4 hours. After filtration, the filtrate was concentratedin vacuo to about ml., ethyl acetate (100 ml.) was added, and theresulting solution was washed with aqueous sodium bicarbonate followedby water. Water (30 ml.) was then added to the ethyl acetate solution,and, with vigorous stirring, lN hydrochloric acid was added drop by dropuntil the pH of the aqueous phase was 2.5. The aqueous layer wasseparated and washed with ether. n-Butanol (150 ml.) was added, and theresulting mixture was evaporated in vacuo until the water was removed.The resulting butanolic solution (40 ml.) was poured into ether (500ml.) whereby an amorphous precipitate separated. It was filtered off,washed with ether, and dried to yield the hydrochloride of the desiredester as a colorless product, with a purity of 83 per cent (determinediodometrically). The l.R.spectrum (KBr) contains bands at:

692 cm.- 976 cm." 1020 cm. I I95 cm. i300 cm. 1370 cm." 1463 cm. 1500cm. 1550 cm.- 1690 cm. l765 cm."

EXAMPLE 3 A Acetoxymethyl a-(o-Nitrophenylsulphenylamino)-Benzylpenicillinate Acetoxymethyl bromide (1.1 ml.) was added withstirring to a solution of a-(o-nitrophenylsulphenylamino)-benzylpenillin(5.0 g.) and triethylamine (1.4 ml.) in methylene chloride (45 ml.). Thereaction mixture was stirred overnight. Thereafter it was washed withwater (25 ml.) aqueous sodium bicarbonate(l0 ml.), and water (10 ml.).The organic phase was dried and evaporated in vacuo to yield a gum whichwas washed by decantation with petroleum ether. B Acetoxymethyla-Aminobenzylpenicillinate Hydrochloride To a solution of the crudeacetoxymethyl a-(onitrophenylsulphenylamino)-benzylpenicillinate,prepared as described under A above, in 50 ml. of ethyl acetate wasadded to solution of 2.2 g. of sodium thiosulphate in 50 ml. of waterwith vigorous stirring. The aqueous phase was separated, extracted withether, neutralized with aqueous sodium bicarbonate (ph 7.5), andextracted twice with ethyl acetate. The organic phase was dried andevaporated in vacuo leaving an oily residue which was dissolved inethanol (25 ml.) by the addition of 2N alcoholic hydrogen chloride (3ml.) with stirring, and precipitated by addition of ether. The amorphousproduct was filtered off and washed with ether, yielding theacetoxymethyl aaminobenzylpenicillinate hydrochloride.

EXAMPLE 4 A Acetoxymethyl a-Benzyloxycarbonylamino-Benzylsuspension wasfiltered and the filtrate evaporated in vacuo. The residue was dissolvedin ethyl acetate and extracted with water, aqueous sodium bicarbonate,and water. The organic phase was dried and evaporated in vacuo to yielda gum which was washed by decantation with petroleum ether. B.Acetoxymethyl Hydrochloride To a solution of orthophosphoric acid(0.98-g.) and potassium dihydrogen phosphate (1.36 g.) in 25 ml. ofwater was added 10 percent palladium on barium sulphate catalyst (6 g.)and the suspension was shaken for 1 hour under hydrogen. A solution ofthe abovementioned gum in 25 ml. of ethyl acetate was added and theresulting mixture shaken in a hydrogen atmosphere for 2 hours at roomtemperature and atmospheric pressure. The catalyst was filtered off, andthe aqueous phase separated and washed with ether. The aqueous phasewas. neutralized(to a pH about 7.5) with aqueous sodium bicarbonate andextracted twice with ethyl acetate. The combined extracts were washedwith water, dried and evaporated in vacuo. Thereafter the resulting oilyresidue was converted to the hydrochloride of acetoxymethyla-aminobenzylpenicillinate as described in Example 33.

EXAMPLE 5 A. Acetoxymethyl D(-)-a-Azidobenzyl Penicillinate PotassiumD(-)-a-azidobenzylpenicillinate (8.26 g.), potassium bicarbonate (1.0g.), and brommethyl acetate (4.] ml.) were refluxed for 1 hour in amixture of acetone (50 ml.) and water (1 ml.).

After cooling, the suspension was filtered, and the filtrate evaporatedin vacuo. The residue was washed repeatedly with petroleum ether toremove excess of bromomethyl acetate. The oily residue was taken up inethyl acetate (50.ml.), and the resulting solution washed with aqueoussodium bicarbonate followed by water. After drying, the solvent wasremoved in vacuo to yield the desired compound as a gum.

B. Acetoxymethyl D(-)-a-Aminobenzylpenicillinate Hydrochloride To asolution of acetoxymethyl D(-)-a-azidobenzylpenicillinate (10.0 g.) inethyl acetate (150 ml.) were added water ml.) and 10 percent palladiumon carbon catalyst (5 g.) in a flask equipped with an efficient stirrer,a gas inlet tube, a gas outlet tube, a combined glass and calomelelectrode, and a burette controlled by an automatic titrator. The systemwas flushed with nitrogen whereafter a stream of hydrogen was bubbledthrough the suspension with stirring, a pH value of 3.0 being maintainedby addition of 1N hydrochloric acid via the automatic titrator. When theconsumption of acid stopped, the flask was flushed witha-Aminobenzylpenicillinate Calculated for C H N O S C 52.55, H 5.26, N7.09,

S 10.80 percent Found: C 52.80, H 5.42, N 6.88, S 10.64 percent EXAMPLE6 Acetoxymethyl a-Amino-Benzylpenicillinate To a stirred suspension ofa-amino-phenylacetyl chloride hydrochloride (340 mg.) and acetoxymethyl6-am-inopenicillinate p-toluenesulphonate (690 mg.) in

methylene chloride (8 ml.) at C. was added a solution of triethylamine(0.50 ml.) in methylene chloride (2 ml.). After reaction for one hour at0 C and for A hour at room temperature, the mixture was evaporated invacuo and treated with ethyl acetate (20 ml.) and water (25 ml.)containing 4N hydrochloric acid (0.35 ml.). After shaking, the layerswere separated, the ethyl acetate was discharged, and the pH of theaqueous solution was adjusted to 7.5 by the addition of saturated sodiumbicarbonate solution (3.5 ml.). The mixture was extracted with ethylacetate, and the organic layer was dried over magnesium sulphate andevaporated to leave the acetoxymethyl penicillin ester as a viscous oil.

The IR. spectrum (CHC1 had bands at 3300 cm. (NH), 1875 cm.- (B-lactam),1760 cm. (ester carbonyl) and 1680 cm.. (amide). The identity of thecompound was demonstrated by conversion of the free base into itshydrochloride andcomparison with an authentic sample:

LR. (KBr):

1765 cm." 1550 cm.

T.L.C.: R 0.51 (butanol-ethano1-H O, 8+2+2) R 0.52 (butanol-aceticacid-H O, 8+2+2) The acetoxymethyl 6-aminopenicillinateptoluenesulphonate used as starting material was prepared in thefollowing manners: Acetoxymethyl 6-aminopenicillinate toluenesulphonateA Acetoxymethyl 6-tritylaminopenicillinate A solution of6-trityaminopenicillanic acid (11.5 g.) in acetone (65 ml.) was cooledto 0 C., triethylamine (4.2 ml.), was added, followed by acetoxymethylbromide (2.45 ml.), and the reaction mixture was kept at 0 C. withstirring for 2 hours and finally at room temperature for 1 hour. Thetriethylammonium bromide (3.5 g.), which precipitated, was removed byfiltration, the evaporated filtrate was treated with ethyl acetate (175ml.), and after 2 washings with cold 2 percent aqueous N aHCO and withice-water, the ethyl acetate layer was dried over'MgSO, to yield, onevaporation, the desired p'ure acetoxymethyl ester as an amorphouspowder.

1687 cm." 1500 cm.

Analysis Calculated for C H N- O S: C 67.77, H 5.87, N 5.27, S 6.04percent Found C 67.73, H 5.91, N 5.22, S 6.00 percent [011 +109.8 (C=2;CHCl Thin-layer chromatography (T.L.C.) on silica gel (Merck HF- showeda pure product.

R 0.71 (butanol-ethanol-H o, 8+2+2) R 0.78 (butanol-acetic acid-H O,8+2+2).

B. Acetoxymethyl 6-Aminopenicillinate, Toluenesulphonate Acetoxymethyl6-aminopenicillinate, (3.71 g.) in acetone (129 ml.) (0.2 percent B 0)was treated with anhydrous p-toluenesulphonic acid (1.21 g.). Afterstanding for 1 15 hours at room temperature, water (0.14 ml.) was added,and precipitation of the ptoluenesulphonate was brought about byaddition of petroleum ether (300 ml.). Filtration and consecutivewashings with acetone-petroleum ether, ethyl acetate and ether left thecrude salt.

After two recrystallizations from acetone-ether, a colorless crystallineproduct was obtained with a melting point of: 132.5-134 C (dec.).calculated for C sH24N20aS2Z C 46.95, H 5.25, N S 13.92 percent Found: C46.84, H 5.17, N 5.86, S 13.79 percent [11],, +127.5(C=1;ethanol). I Thepurity was further established by T.L.C.

R 0.51 (butanol-ethanol-l-QO, 8+2+2) R 0.53 (butanol-CH COOHH O, 8+2+2)lodometric assay showed 99 percent purity using 6- aminopenicillanicacid as a standard. Acetoxymethyl-6-Aminopenicil1inate,toluenesulphonate 6-Aminopenicillanic acid (4.32 g.), dispersed inacetone ml.), was treated with triethylamine (6.3 ml.) with stirring at0 C.. Acetoxymethyl bromide (3.92 ml.) was added dropwise, and themixture was kept with stirring at room temperature for 4 hours.Triethylammonium bromide (3.5 g.), which formed, was removed byfiltration, and was evapdrated to dryness in vacuo, then treated withthe filtrate/ethyl acetate (140 ml.) and cold 2 percent sodiumbicarbonate (l10m1.). After shaking and separation, the ethyl acetatesolution was further shaken with icewater and dried over magnesiumsulphate.

On evaporation, 5.2 g. of product were obtained The crude ester,dissolved in acetone (200 ml.), was treated with anhydrousp-toluenesulphonic acid (3.5 g.) and the p-toluenesulphonate wasprecipitated by added ether (750 ml.). It was collected by filtrationand washed with ethyl acetate and ether to yield the desired compound.

The complete identity with the product obtained above was demonstratedby comparing melting points, I.R.spectra and T.L.C. data. IR. (KBr)showed strong bands at:

1785 cm." 1758 cm. 1379 cm. 1325 cm." 1225 cm." 1170 cm. 1123 cm." 1030cm. 1006 cm. 972 cm."

811 cm. 681 cm."

EXAMPLE 7 Acetoxymethyl a-Azido-Benzylpenicillinate Acetoxymethyl-aminopenicillanate ptoluenesulphonate (580 mg.) was added to a mixtureof ethyl acetate (50 ml.) and 2 percent aqueous sodium bicarbonate(50m1.). a-Azido-a-phenylacetyl chloride (250 mg.) dissolved in benzene(5 ml.) was added to the mixture with vigorous stirring at 0 C.. After1; hour, the ethyl acetate layer was shaken with ice-water, died overmagnesium sulphate and evaporated in vacuo to give the desired compound.

IR. (KBr):

2125 cm.- (azido group) 1780 cm. (B-lactam) 1765 cm." (ester carbonyl)1690 cm." (amide).

EXAMPLE 8 Acetoxymethyl a-Azido-Benzylpenicillinate Acetoxymethyl6-aminopenicillanate, ptoluenesulphonate (600 mg.) was shaken with amixture of (acetate 935 ml). and cold 2 percent sodium bicarbonate (17ml.), the layers were separated, and the ethyl acetate solution wasfurther shaken with icewater (20 ml). then dried over magnesiumsulphate, and evaporated to leave the free acetoxymethyl ester (360mg.). This was dissolved in methylene chloride (8 ml.),N,N'-dicyclohexylcarbodiimide (255 mg.) was added, and the resultingsolution was added quickly with stirring to a-azido-phenylaceticacid'(220 mg.) in N ,N-dimethylformamide (2.5 ml.) at 0 C.. The stirringwas continued for 1 hour at room temperature, and the mixture wasfiltered over diatomaceous earth Dicalite (Registered Trade Mark).

The volume of the filtrate was reduced in vacuo, ethyl acetate was added(25 ml.), and after shaking with 0.01 N hydrochloric acid and with 2percent sodium bicarbonate, the ethyl acetate layer was dried overmagnesium sulphate. Removal of the solvent at room temperature gave aproduct which provided identical with the compound obtained according tothe procedure of Example 7.

EXAMPLE 9 A. Propionyloxymethyl D()-a-Azidobenzylpenicillinate Thiscompound was prepared analogously to the pivaloyloxymethylD(-)-a-azidobenzyl penicillinate described in Example 12A, usingchloromethyl propionate as the halomethyl ester reagent. B.Propionyloxymethyl D(-)-a-Aminobenzylpenicillinate, Hydrochloride Thecompound was prepared by catalytic hydrogenation of propionyloxymethylD(-)-a-azidobenzylpenicillinate in the same way as described in Example12B.

The desired compound was obtained as a colorless, amorphous powder, andhad a purity of 90 percent determined iodometrically. T.R.(KBr):1780(shoulder), 1764, and 1690 cm. N.M.R. (D 0): Signals at =7.94 (s), 6.25(d; J=l cps), 5.96 (s), 5.71 (s), 5.00 (s), 2.88 (m;j=7 cps), 1.85 (s),and 1.53 (t; j=7 cps), TMS being used as external reference. [a]:+191C(c=1inH 0).

EXAMPLE A. Butyryloxymethy D(-)-a-Azibobenzylpenicillinate The compoundwas prepared from D(-)-aazidobenzylpenicillin potassium salt andchloromethyl butyrate in a manner analogous with the preparation ofpivaloyloxymethyl D(-)-a-azidobenzylpenicillinate described in Example12A.

B. ButyryloxymethylD(-)-a-Aminobenzylpenicil1inate, Hydrochloride Thecompound was prepared by catalytic hydrogenation of butyryloxymethylD(-)-a-azidobenzylpenicillinate, using the method described in Example128, and obtained as a colorless, amorphous powder.

The purity of the compound was determined iodometrically to be 90.2percent.

1.R. (KBr): Bands at 1780-1775 (shoulder), 1763, and 1688 cm.'. N.M.R.(D 0: Signals at 6 7.94 (s), 6.25 (d;.l=1.5 cps), 5.97 (s), 5.73 (s),5.00 (s), 2.82 (t; J=percent 5 cps), 2.04 (m; 7.5 cps), 1.83 (s), and1.34 (t; J=7.5 cps), TMS was used as external reference. [a],, +197 C(c=1 in H O).

EXAMPLE 1 1 A. lsobutyryloxymethyl linate This compound was preparedfrom potassium D(-)- a-azidobenzylpenicillinate and chloromethylisobutyrate in the same manner as described in Example 12A for thepreparation of pivaloyloxymethyl D(-)-aazidobenzylpenicillinate. B.lsobutyryloxymethyl linate The compound was obtained as a colorless,amorphous powder by the catalytic hydrogenation of isobutyryloxymethylD(-)-a-azidobenzylpenicillinate, using the method described in Example12B. The compound had a purity of 92.6 percent determinediodometrically. IR. (KBr): Bands at 1780 (shoulder), 1760-1755, and 1690cm. N.M.R. (D 0): Signals at 8 7.94 (s), 6.27 (d; J=l.5 cps), 5.98 (s),5.71 (s), 5.01 (s), 3.09 (m; J=7 cps), 1.82 (s) 1.57 (d; J=7 cps) TMSwas used as external reference. [a] :+l96 (c=1 in H O).

EXAMPLE 12 A. Pivaloyloxymethyl D(-)-oz-Azidobenzylpenicillinate To asuspension of potassium D(-)-a-azidobenzylpenicillinate (4.14 g.) andpotassium bicarbonate (1.5 g.) in acetone (100 ml.) and 10 percentaqueous sodium iodide (2ml.) chloromethyl pivalate (2.7 ml.) was addedand the mixture refluxed for 2 hours. After cooling, the suspension wasfiltered and the filtrate evaporated to dryness in vacuo. The remainingresidue was washed repeatedly by decantation with petroleum ether toremove unreacted chloromethyl pivalate. The oily residue was taken up inethyl acetate (100 ml.), and the resulting solution washed with aqueoussodium bicarbonate and water, dried and evaporated in vacuo to yield thedesired compound as a yellowish gum, which crystallized from ether. M.p.l14-115 C. [a] +42 (c=1, CHCI The LR. Spectrum (KBr) con-D(-)-a-Azidobenzylpenicil- D(-)-a-aminobenzylpeniciltained strong bandsat: 2130, 1786, 1760, 1700, I530,

1225,1110, and 973 cm.

Pivaloyloxymethyl D(-)-a-Aminobenzylpenicillinate, Hydrochloride To asolution of pivaloyloxymethyl D(-)-aazidobenzylpenicillinate (preparedas described above) in ethyl acetate ml.) a 0.2 M phosphate buffer (pH2.2 (75 ml.) and 10 percent palladium on carbon catalyst (4g.) wereadded, and the mixture'was shaken in a hydrogen atmosphere for 2 hoursat room temperature. The catalystwas filtered off, washed with ethylacetate (25 ml.) and phosphate buffer (25 ml.), and the phases of thefiltrate were separated. The aqueous phase was washed with ether,neutralized (pl-l 6.5-7.0) with aqueous sodium bicarbonate, andextracted with ethyl acetate (2 X75 ml). To the combined extracts, water(75 ml.) was added, and the pH adjusted to 2.5 with ,1 N hydrochloricacid. The aqueous layer was separated, the organic phase extracted withwater (25 ml.), and the combined extracts were washed with ether, andfreeze-dried. The desired compound was obtained as a colorless,amorphous powder, soluble in water, methanol and ethanol. TheIR-spectrum (KBr) contained bands at 1780 (shoulder), 1765-1775, and1960 emf.

The NMR-spectrum (D showed signal at 8=7.94 (s); 6.20 (m); 5.94 (s).5.78 (s), 4.96 (s) 179 (s), and 1.55 (s) TMS being used as externalreference.

[11], +1 95 (c+1 in H from isopropanol The purity of the compound wasdetermined iodometrically to be 91 percent. A crystalline hydrochloridewas obtained from isoporpanol with a melting point of 155l56 C. (dec.).

[011 +l96 (c=1 in 11 0).

EXAMPLE 13 Pivaloyloxymethyl 6-Aminopenicillanate, P- ToluenesulphonateTo a stirredsolution of triethylammonium 6-tritylaminopenicillanate (5.6g.) in acetone (25 ml.) at 0 C was added triethylamine (1.08 ml.)followed by chloromethyl pivalate (1.6 ml.) and triethylammonium iodide(2.3 g.) Stirring was continued at 0 C for A hour and at roomtemperature for 20 hours. The precipitate of triethylammonium chloridewas removed by filtration, and the evaporated filtrate was treated withethyl acetate (75 ml.). After shaking with cold 1 percent aqueous NaHCOand with ice-water, the ethyl acetate layer was separated, dried overMgSO, and evaporated in vacuo to leave the pivaloyloxymethyl ester of6-tritylaminopenicillanic acid as an amorphous powder. To the crudeester, dissolved in ethyl acetate (40 ml.) was added a solution ofp-toluenesulphonic acid, hydrate (1.90 g.) in ethyl acetate (20 ml.) at0 C. After additional stirring for 2 hours at room temperature, theprecipitate of the desired tosylate was collected on a filter and washedwith ethyl acetate and ether.

Recrystallization from acetone-ether afforded the pure crystallinecompound with a melting point of 13839bL C. (dec.).

Analysis C H- N O S, requires a C 50.18, H 6.01, N 5.57, S 12.76 percentFound: C 50.46, H 6.15S 12.57 percent 1.R. (KBr) had strong bands at1795, 1768, 1750, 1545, 1485, 1465, 1375, 1365, 1315, 1210, 1160,1115,1030, 1010, 980, 815 and 680 emf.

Iodometric assay, using 6-aminopenicillanic acid asreference, showed 99percent purity.

The purity of the compound was further demonstrated by thin-layerchromatography (T.L.C.) on silica gel (Merck, HF R 0.66(n-butanol-ethano1-H O,4+ 1+1) R 0.12 (cyclohexane-ethyl acetate, 1+1).

EXAMPLE l4 Pivaloyloxymethyl 6-aminopenicillanate, ptoluenesulphonate.

-Aminopenicillanic acid (4.32 g.), dispersed in acetone (140 m1.) at 0C, was treated with triethylamine (6.4 ml.). While maintaining stirringat 0 C, chlormethyl pivalate (5.9 m1.) and triethylammonium iodide (4.6g.) were added, and the stirred mixture was left for 20 hours at roomtemperature. Triethylammonium chloride (2.9 g.) was removed byfiltration and the evaporated filtrate was worked up according to theprocedure described in Example 13 to yield the crude pivaloyloxymethylester. This was taken up in ethyl acetate (45 m1.). Treatment withanhydrous ptoluenesulphonic acid (3.4 g.) in ethyl acetate 50 ml.)caused immediate precipitation of the crystalline tosylate which wasfiltered off and washed with ethyl acetate and ether, yielding a purecolorless material which in every respect proved identical with theanalytically pure compound from Example 13.

EXAMPLE l5 Pivaloyloxymethyl 6-aminopenicillanate, toluenesulphonate Adispersion of 6-aminopenicillanic acid (2.16 g.) in acetone (ml.) at 0 Cwas treated with triethylamine (3.22 ml.) followed by bromomethylpivalate (3.92 g.) (b.p.: 505 1 C at 10 mm Hg.) prepared from pivaloylbromide and paraformaldehyde by a method analogous to the preparation ofchloromethyl pivalate described in .1.A.C.S. 89,5442 1967).

After stirring for 20 hours at room temperature, the precipitate oftriethylammonium bromide 1.63 g.) was collected on a filter, and theevaporated filtrate was worked up according to the procedure describedin Example 13. The crude pivaloyloxymethyl ester was dissolved in ethylacetate (45 ml.), and crystallization of its pure tosylate was brought.about by addition of anhydrous p-toluenesulphonic acid (1.72 g.) inethyl acetate (25 ml.). The product, which was isolated after filtrationand washings with ethyl acetate and ether, showed complete identity withthat of Example 13.

EXAMPLE 16 Pivaloyloxymethyl 6-aminopenicillanate, ptoluenesulphonate To6 -aminopenicillanic acid (2.16 g.) dispersed in acetone (60 ml.) at 0 Cwas added triethylamine 1.8 ml.) followed by an acetone solution (10ml.) of pivaloyloxymethyl p-toluenesulphonate (3.2 g.). The mixture wasstirred for 1% hour at 0 C. and for 20 hours at room temperature. The 1resulting solution was evaporated in vacuo, and the residue was workedup by the method applied in Example 13. To the crude ester in ethylacetate ml.) was added anhydrous ptoluenesulphonic acid 1.7 g.) in ethylacetate (25 ml.). The precipitated crystalline salt after filtration andwashings with ethyl acetate proved identical with the compound preparedaccording to the previous Example 13. 33.542 The starting material,pivaloyloxymethyl p l toluenesulphonate, was prepared by reacting thesilver salt of p-toluenesulphonic acid with chloromethyl pivalate in dryacetonitrile for 4 days at room temperature. It was obtained with amelting point of 44-33.5 C.

Analysis C H O S requires: C 54.53, H 6.33, S 11.2. percent Found: C54.73, H 6.31, S 11.09 percent EXAMPLE 17 Pivaloyloxymethyl6-aminopenicillanate, toluenesulphonate To a dispersion of6-aminopenicillanic acid (2.16 g.) and potassium bicarbonate (1.0 g.) inacetone (70 ml.) were added chloromethyl pivalate (1.56 ml.) andpotassium iodide (280 mg.). The mixture was stirred and refluxed for 5hours. Solid material was filtered off, and the acetone solution wasevaporated in vacuo. The residue was triturated with ethyl acetate(80m1.), and, after filtration, the cooled ethyl acetate solution wastreated with anhydrous p-toluenesulphonic acid (1.72 g.) in ethylacetate (40 ml.). The precipitated tosylate was collected on a filterand washed with ethyl acetate and ether. Melting point. l.R.spectrum andT.L.C. data of the colorless crystalline product established theidentity with the compound obtained according to previous examples.

EXAMPLE 18 Pivaloyloxymethyl 6-aminopenicillanate, hydrochloride To asolution of PCl 5 (2.1 g.) in 20 ml. of dry, alcohol free chloroform wasadded 2.26 ml. of dry quinoline with stirring. The resulting suspensionwas cooled to 30 C, and 4.0 g. of pivaloyloxymethyl benzylpenicillinatewere added. After 30 minutes stirring at 5 C, to -l, C, the solution wascooled to -30 C and 6.7 ml. of dry n-propanol were added in one portion.The temperature rose to l0 C to 15 C. The reaction temperature wasraised to 0 C during 15 minutes and kept at this temperature for 30minutes, whereafter the solution was added to an ice-cold mixture ofwater (25 ml.) and hexane (40 ml.) with stirring. The aqueous phase wasseparated and the organic phase extracted three times with 25 ml. ofice-cold 1 N HCl. The combined aqueous phases were stirred at 0ml. Cwith 90 mml. of ethyl acetate, the pH being adjusted to 7.5 with NaHCOThe organic phase was separated, dried and evaporated in vacuo to leavean oil which was dissolved in 50 m1. of isopropanol at 0 C. By additionof 1.75 ml. of an 8 N dry solution of hydrochloric acid in isopropanolwith stirring, the desired hydrochloride was precipitated. It wasfiltered and subsequently washed with isopropanol and ether to give apure product with a melting point of l56-160 C. (dec.). AnalysisCalculated for C H ClN O S: C 45.84, H 6.32, Cl 9.66, N 7.63 S 8.74percent Found: C 45.60, H 6.39, CI 9.76, N 7.54, S 8.83 percent [111+183(c=1, 0.1 N HCl). l.R. (KBr) had characteristic strong carbonylbands at 1790,1767 and 1756 cm.'.

The starting material could be prepared in the following manner;Pivaloyloxymethyl benzylpenicillinate To a suspension of potassiumbenzylpenicillinate (19.0 g.) in 200 ml. of acetone was addedchloromethyl pivalate (8.3 m1.) followed by a solution of sodium iodide(1.25 g.) in water (5 ml.) The mixture was refluxed for 5 hours. Aftercooling, the potassium chloride was removed by filtration. By addingwater to the filtrate, the desired compound was obtained as a colorlesscrystalline product with a melting point of l l4-1 15 C. AnalysisCalculated for CggHggNgOaSi C5831. H N percent Found: C 58.82, H 6.33, N6.28 percent [a] +236(c=1; methanol). Thin-layer chromatography onsilica gel (Merck HF showed a pure product. R 0.45(Cyclohexane-ethylacetate, 1+1) R 0.86 (Butanol-ethanol-H O, 4+1+l In a manner analogouswith that described in the procedure above, the pivaloyloxymethyl 6-aminopenicillanate was prepared by substituting phenoxymethyl penicillinfor benzylpenicillin.

EXAMPLE 19 Pivaloyloxymethyl 6-aminopenicillanate, p-

toluenesulphonate (24.0 g.), suspended in ethyl acetate (1.1 l), wastreated with 2 percent aqueous sodium bicarbonate (760 ml.) withvigorous stirring. The layers were separated, and the ethyl acetatesolution was shaken thoroughly with ice-water (600 ml.) with 2 percentsodium bicarbonate added (25 ml.).

The ethyl acetate layer was dried over anhydrous magnesium sulphate,filtered and evaporated in vacuo. The residue was treated with petroleumether (200 ml.)(b.p. 50 C.) and crystallization took place on stirringfor 2% hours, yielding the analytically pure ester with a melting pointof 65 C.

Analysis C,.,H N O S requires: C 50.90, H 6.71, N 8.48, S9,71 percentFound: C 51.15, H 6.77, N 8.36, S 9.63

l.R. (KBr)showed characteristic bands at 3400, 1780, i

and 1750cm..

EXAMPLE 20 EXAMPLE 21 Pivaloyloxymethyl D(-)a-aminobenzylpenicillinate,hydrochloride To a solution of potassium N-[l-methyl-Z-carbethoxy-vinyl]-D-(-)-a-amino-wphenylacetate (155.2 g.; 0.5moleof the hemihydrate) in ethyl acetate (2 l.),' N- methylmorpholine(2.5 ml.) and isobutyl chloroformate (70 ml.) were added at l C. withstirring. Potassium chloride separated immediately, and the mixture waskept at 1 5 C for 6 minutes. Then an icecold solution ofpivaloyloxymethyl o-aminopenicillanate in ethyl acetate (1].) preparedfrom 251.3 g of the crystalline p-toluenesulphonate of this compound wasadded with stirring, the temperature being kept between --l4 C. and 12C. during the acyl ation. After stirring for another minutes at lowtemperature, the cooling bath was removed, and the mixture stirred atroom temperature for 30 minutes. Thereafter the mixture was extractedwith 0.5 M aqueous sodium sodium bicarbonate (500 ml.), and washed withwater (2 X 250 ml.). The organic phase was dried, and the solventremoved in vacuo. The yellow oily residue thus obtained was dissolved inacetone (1 1.); water (0.9 l.) was added, and 4 N hydrochloric acid wasadded drop by drop to the mixture with vigorous stirring. A pH- value of2.5 was maintained in the mixture during the hydrolysis by using anautomatic titrator. The reaction was finished when the consumption ofhydrochloric acid ceased after addition of 100-110 ml. (80-88 per centof the theoretical amount). Acetone was removed from the mixture byevaporation in vacuo (bath-temperature about 35 C.), and the remainingaqueous phase was extracted several times with ethyl acetate. Afterseparation of the aqueous layer, the combined ethyl acetate extractswere diluted with petroleum ether (800 ml.) and extracted with water (pH3; 200 ml.). To the combined aqueous phases (ca. 1.2 l) sodium chloride(240 g.) was added, and the mixture was shaken vigorously whereafter ayellowish organic layer was separated. The aqueous phase was furtherextracted with ethylacetate (200 ml.), and the combined organic phaseswere dried over magnesium sulphate, filtered, ml.), isopropanol (800ml.) was added to the filtrate. After concentration of the solutionunder reduced pressure to about half the volume (bath-temperature about35 C.), another 800 ml. of isopropanol were added, and the mixture wasconcentrated in vacuo to about 600-800 ml. Crystalline materialseparated from the mixture, which was stirred for 1 hour at. roomtemperature and was kept in the refrigerator overnight. The crystallinematerial was fil-.

tered off, washed with ice-cold isopropanol (100-ml.), and ether (2 X100 ml), and dried at room temperature to yield the desired compound ascolorless crystals, m.p. l55-l56C. (dec.).

EXAMPLES 22-26 By the method of Example 15, acetoxymethyl 6-aminopenicillanate, propionyloxymethyl 6- aminopenicillanate,butyroxymethyl 6-aminopenicillanate, isobutyryloxymethyl6-aminopenicillanate, and octanoyloxymethyl -aminopenicillanate wereprepared by substituting the corresponding bromomethyl acylate for thebromomethyl pivalate.

EXAMPLE 27 A. 2-Ethyl-butyroyloxymethyl penicillinate The compound wasprepared from potassium D(-)- oz-azidobenzylpenicillinate andchloromethyl 2-ethylbutyrate in the same manner as described for thepreparation of pivaloyloxymethyl D(-)-a-azidobenzylpenicillinate inExample 12A.

B. 2Ethyl-butyroyloxymethyl D(-)-a-aminobenzylpenicillinate,hydrochloride colorless This compound was prepared by the catalytichydrogenation of 2-ethylbutyroyloxymethylD(.-')-aazidobenzylpenicillinate using the method described in Example123, and was obtained as a colorless amorphous powder having a purity of86.4 percent determined iodonetrically.

IR. (KBr): Bands at 1780(shoulder), 1762, and 1690 cm.. N.M.R.( 0):Signals at 7.92(s), 6.22(m'), 5.93(s), 5.77(s), 4.96(s), 2.68(m; J=7cps), l.95(m; J=7 cps), l.82(s), and l.25(t; J=7 cps), TMS was used asexternal reference.

EXAMPLE 28 D(-)-a-azidobenzyl- A. BenzoyloxymethylD(-)-a-azidobenzylpenicillinate To a suspension of potassiumD(-)-oz-azidobenzylpenicillinate (4.14 g.) in a mixture of acetone 100ml.) and 10 percent aqueous sodium iodide (2 ml.), chloromethyl benzoate(2.5 g.) was added, and the mixture refluxed for 6 hours. After cooling,the suspension was filtered and the filtrate evaporated to dryness invacuo. The residue was washed with light petroleum to remove excess ofchloromethyl benzoate, and thereafter dissolved in ethyl acetate (50ml.). The solution was washed with aqueous sodium bicarbonate followedby water, dried, and evaporated in vacuo to yield thedesired compound asa gum.

B. Benzoyloxymethyl D(-)a-aminobenzylpenicillinate, hydrochloride To asolution of benzoyloxymethyl D(-)-aazidobenzylpenicillinate (5.0 g.) inethyl acetate ml.) was added water (50 ml.) and 10 percent palladium oncarbon catalyst (3 g.) in a flask equipped with an efficient stirrer, agas inlet tube, a gas outlet tube, a glass-calomel combinationelectrode, and a burette controlled by an automatic titrator. The systemwas flushed with nitrogen whereafter a stream of hydrogen was bubbled.through the suspension with stirring, a pH value of 3.0 of the aqueousphase being maintained by addition of l N hydrochloric acid via theautomatic titrator. When the consumption of acid stopped, the flask wasflushed with nitrogen until all hydrogen was removed, and the catalystwas filtered off. The biphasic filtrate was separated and the aqueousphase washed with ether and freeze-dried. The desired compound wasobtained as a colorless amorphous powder, easily soluble in water.

[04], 75(F1, H20). The N,M,R.-spectrum (D 0) showed signals at 67.93(m)(l0 H), 6.29(m)(2 l-l), about 5.84(m)(3 H),

4.90(s)(l H), l.66(3 H) and 1.54 (3 H), TMS being used as externalreference.

The purity of the compound was determined iodometrically to be 92percent.

EXAMPLE 29 A. 2-Methylbenzoyloxymethyl penicillinate This compound wasD( )-a-azidobenzylbenzoate was prepared from paraformaldehyde and l-EXAMPLE 30 A. 2,6-Dimethylbenzoyloxymethyl azidobenzoylpenicillinate Thecompound was prepared from potassium D(-)- a-azidobenzylpencillinate andchloromethyl 2,6- dimethylbenzoate as described in Example 28A. Thechloromethyl 2,6-dimethylbenzoate was prepared from paraformaldehyde and2,6-dimethylbenzoylchloride benzoate described in J.A.C.S. 43,662( 1921B. 2,6-Dimethylbenzoyloxymethyl D(-)-aaminobenzylpenicillinate,hydrochloride.

This compound was prepared by catalytic hydrogenation of2,6-dimethylbenzoyloxymethyl D(-)- a-azidobenzylpenicillinate in thesame way as described in Example 288. The desired compound was obtainedas a colorless, amorphous powder with a purity of 85 percent determinediodometrically.

EXAMPLES 31-53 Using the method described in Example 28A and B, butsubstituting the XC1-l OCO(CI-I ),,A defined in the Table IV below forthe chloromethyl benzoate, the corresponding esters ofD(-)-a-aminobenzylpenicillin are obtained.

TABLE IV ex no X n A Isolated as 31 Cl 2,6-dimethoxyphenyl hydrochloride32 Cl 0 2-methyl-6-chlorophenyl hydrochloride 33 Br 03-trifluoromethylphenyl hydrobromide 34 Cl 0 Z-nitrophenyl hydrochloride35 Cl 0 Z-methylthiophenyl citrate 36 Br 0 Z-thienyl hydrobromide 37 Br0 2-furyl hydrobromide 38 Br 2 2-furyl hydrobromide 39 Cl 0 Z-quinolylhydrochloride 40 Cl 1 Z-thienyl hydrochloride 41 Br 1 phenyl tartrate 42Cl 3 p-tolyl maleate 43 Cl 0 3 -pyridyl hydroiodide 44 Cl 0 4-pyridylhydrochloride 45 Cl 0 l-naphthyl hydrochloride 46 Cl 0 Z-naphthylhydrochloride 47 Cl 0 pyrazinyl hydrochloride 48 Cl 1 cyclohcxylhydrochloride 49 Cl 1 2-methylcyclopentyl hydrochloride 50 Cl 4Z-methylcyclopentyl hydrochloride 51 Cl 0 l-bicyclo[2.2.2]octy1 sulphate52 Cl 0 l-adamantyl hydrochloride 53 Cl 0 cyclohexen-Z-yl hydrochlorideSome of the halomethyl esters used are new compounds, which as mentionedbefore can be prepared from paraformaldehyde and the corresponding acidhalide analogous to the preparation of chloromethyl benzoate describedin .I.A.C.S. 43, 660( 1921 The salts obtained according to the lastcolumn of Table IV correspond to the acids used to maintain the acidityduring the hydrogenation. When e.g. hydrochloric acid is used ahydrochloride is obtained, whereas if e.g. citric acid is used a citrateis obtained.

EXAMPLE 54 Benzoylxymethyl hydrochloride D( )-a-aminobenzylpenicillin(3.5 g.) and triethylamine (1.42 ml.) were mixed with acetone containing1 per cent of water ml.). To the resulting solution was added potassiumbicarbonate (l g.) and bromomethyl benzoate (4.0 g.) whereafter themixture was stirred at room temperature for 4 hours. After filtration,the filtrate was concentrated in vacuo to about 15 ml., ethyl acetateml.) was added, and the resulting solution was washed with aqueoussodium bicarbonate followed by water. Water (30 ml.) was then added tothe ethyl acetate solution, and with vigorous stirring, 1' Nhydrochloric acid was added drop by drop until the pH of the aqueousphase was 2.5. The aqueous layer was separated and washed with ether;nbutanol( ml.) was added, and the resulting mixture was evaporated invacuo until the water was removed. The resulting butanolic solution (40ml.) was poured into ether (500 ml.) whereby an amorphous precipitateseparated. It was filtered off, washed with ether and dried to yield thehydrochloride of the desired ester as a colorless product, identicalwith the compound of Example 28B.

By the methods of Examples 19, 20 and 54 the compounds of Table IV canalso be obtained.

D( -)-a-aminobenzylpenicillinate EXAMPLE 55 Mixture containingpivaloyloxymethyl aminobenzylpenicillinate, hydrochloride Aluminummonostearate (75 g.) was dissolved in modified coconut oil (4300 g.) byheating to 140 C. After cooling, flavor (15 g.) was added. Saccharose(500 g.) and pivaloyloxymethyl D(-)-a-amino benzylpenicillinatehydrochloride g.) were mixed, reduced by milling to a particle size ofabout 10 micron and mixed with the oily solution by means of a highspeed stirrer.

A mixture was obtained containing 35 mg. of pivaloyloxymethylD(-)-a-aminobenzylpenicillinate hydrochloride per ml.

What we claim is:

1. A compound selected from the group consisting of esters ofa-aminobenzylpenicillin and pharmaceutically acceptable salts thereof ofthe formula:

S CH3 v Q-CH-CONH-CH-Cfi c i I l cH3 NH; O-N- HCOCH 0CO(CH2)uA wherein nis an integer from 0 to 5, A is alkyl of one to 3. Acetoxymethyla-aminobenzylpenicillinate and six carbon atoms, cyclopentyl,methylcyclopentyl, non-toxic acid salts thereof. cyclohexyl,l-bicyclo(2.2.2)octyl, cyclopentenyl, 4. AcetoxymethylD(-)-a-aminobenzylpenicillinate cyclohexenyl, phenyl, methylphenyl,methoxyphenyl, an on-toxic acid salts thereof. di ethyl henyl, di th hl, 5. Pivaloyloxymethyl a-aminobenzylpenicillinatetrifluoromethylphenyl, methylchlorophenyl, and Hormone add Saltsthereofmethylthiophenyl, nitrophenyl, furyl, thienyl, quinolyl,Pivaloyloxymethyl y p pyridyl, or pyrazinyl linate and non-toxic acidsalts thereof.

2. A compound according to claim 1 wherein n is 0 PivaloyloxymethylD(')'a'aminobenzylpenicil' and A is alkyl of one to six carbon atoms.lmate hydrochlonde-

2. A compound according to claim 1 wherein n is 0 and A is alkyl of oneto six carbon atoms.
 3. Acetoxymethyl Alpha -aminobenzylpenicillinateand non-toxic acid salts thereof.
 4. Acetoxymethyl D(-)- Alpha-aminobenzylpenicillinate and non-toxic acid salts thereof. 5.Pivaloyloxymethyl Alpha -aminobenzylpenicillinate and non-toxic acidsalts thereof.
 6. Pivaloyloxymethyl D(-)- Alpha-aminobenzylpenicillinate and non-toxic acid salts thereof. 7.Pivaloyloxymethyl D(-)- Alpha -aminobenzylpenicillinate hydrochloride.